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Quality Assurance in Bacteriology and Immunology 2002 PDF
Quality Assurance in Bacteriology and Immunology 2002 PDF
28
The views expressed in this publication are those of the author and do not
necessarily reflect the decisions or stated policy of the World Health
Organization; however they focus on issues that have been recognized by the
Organization and Member States as being of high priority.
Printed in India
WHO Regional Publication, South-East Asia Series No.28
Quality
Assurance
IN
Sudarshan Kumari
Regional Adviser
Blood Safety and Clinical Technology
Rajesh Bhatia
Short-term Consultant
Blood Safety and Clinical Technology
1. INTRODUCTION ...................................................................... 1
1.1 Benefits of quality ............................................................. 2
1.2 Continuous quality improvement ....................................... 3
1.3 Users’ Perception of health laboratory services .................... 4
1.4 Good laboratory practices.................................................. 5
1.5 International standards organization.................................... 6
1.6 Traceability....................................................................... 7
8. VALIDATION........................................................................... 54
8.1 Definition ....................................................................... 54
8.2 Process of validation........................................................ 55
9. ASSESSMENT OF QUALITY...................................................... 57
9.1 Monitoring ..................................................................... 58
9.2 Assessment of quality....................................................... 58
9.3 Organization of EQAS...................................................... 66
9.4 Scoring system in EQA..................................................... 69
9.5 Internal quality assessment............................................... 72
INDEX..........................................................................................177
mm millimeter
NCCLS National Committee on Clinical Laboratory Services
(USA)
o
C degree centrigrade
ONPG o-nitrophenyl-β-D-galactopyranoside
OPD outdoor patient department
p.a. pro analysis
PHA passive haemagglutination
p.p.a. purissimum pro analysis
QA quality assurance
QC quality control
RBCs red blood cells
RPR rapid plasma reagin
SOP standard operating procedures
STA standard tube agglutination test
TQM total quality management
VDRL venereal diseases research laboratory
XLD xylose-lysine-desoxycholate (agar)
ZN ziehl neelsen (staining)
Accuracy
The degree to which a measurement or an estimate based on
measurements, represents the true value of the attribute that is being
measured.
Accession list
This list records all specimens that are received in the laboratory for
processing.
Accreditation
This is the process of inspection of laboratories and their licensing by
a third party to ensure conformity to predefined criteria.
Audit
An examination or review that establishes the extent to which a
condition, process or performance conforms to predetermined
standards or criteria.
Bias
Deviation of results or inferences from the truth, or processes leading
to such deviation.
Coefficient of Variation
The standard deviation expressed as a percentage of the mean
Control Serum
Serum with known concentration used to measure the accuracy and
precision and sensitivity and specificity of a procedure. They are used
to determine, verify and document performance.
Dilution
The ratio of the volume of serum or other medium of the total
volume produced by combining it with diluent. The dilution 1:10
denotes one part serum plus nine parts diluent for a total volume of
10 parts. A dilution of 1:1 indicates no dilution. The same notation is
used in areas other than serology to indicate volume of material to
volume of diluent. So the two methods must be carefully
differentiated.
Efficiency
The ability of a test to give a positive result on positives and negative
results on negatives.
Error
The difference between an observed or measured value and the best
obtainable estimates of its true value.
Limit of detection
The limit of detection is the lowest level of analyte that can be
detected, but not necessarily determined in a quantitative fashion,
using a specific method under the required experimental conditions.
Precision
The closeness of the relationship between replicate determinations
with no regard for bias error. Freedom from variation.
Reproducibility. An inverse function of variance; greater the precision
smaller is the variance (ISO/DIS 3534-1; 1990,3.14 )
Predictive values
The predictive value of a positive test result is defined as the
percentage of positive results that are true positives in a given
situation. It indicates the probability that a patient with a positive test
result has, in fact, the disease in question. The predictive value of a
negative test result is the percentage of negative results that are truly
negative for the same given situation. The predictive value of test
results is directly dependent on the prevalence of the condition in the
population being tested. This also implies that predictive values are
not constant but change with the prevalence of the situation.
Probability
Quantitative measure of chance. The ratio of the number of
outcomes that produce a given event to the total number of possible
outcomes.
Range
The difference between the maximum and minimum of a set of
values.
Reference Material
A material of a substance with values of measurable quantities
sufficiently well established to be used for calibration of a
measurement system, the assessment of measurement procedures or
for assigning values to materials ( ISO Guide 30:1981, 2.1).
Sample
A subset or group of objects or things selected from a larger set or
population. Usually information obtained from samples is used to
make inferences about the population.
Sensitivity
Diagnostic sensitivity: The incidence of true positive results obtained
when a test is used for patients known to have the disease or
condition.
True Positive
Sensitivity = x 100
True Positive + False Negative
Specificity
A measure of the ability of a test to indicate the absence of a
component in a specimen when it is truly negative for that
component, or a measure of the ability of a test to measure
accurately one component in a specimen without interference by
other components.
True Negative
(2) Specificity = x 100
True Negative + False Positive
Titer
The reciprocal of the highest dilution of a constituent which leads to a
positive reaction. For example, if a serum produces a positive
reaction in the tube which contains 1:80 dilution but not in the next
higher dilution, then it has a titer of the reciprocal of 1:80, or 80. The
dilution is 1:80; the titer is 80. A titer of 80 means that the serum is
positive in a 1:80 dilution.
Validation
Confirmation by examination and provision of evidence that specific
requirements for specific intended use are met.
Initially the quality of the final product was all that was
looked at. This soon changed to include the processes that
gave the product. From that a broader view of quality
developed and concepts such as good laboratory practices
(GLP) arose followed by customer satisfaction. This has now
been taken further to the approach of total quality
management in which the focus is on the satisfaction of
customers, suppliers, staff and society, and even
consideration of environmental issues.
ISO No Refers to
ISO 9000 series Design/development,
(9000, 9001, 9002, 9003, 9004 production, installation,
etc) servicing and final inspection
and testing as applied to
manufacturing processes
ISO Guide 25 Performance of objective
(ISO Guide 17025) measurements, use of
reference material and
calibration as well as test
methodology
1.6 TRACEABILITY
Traceability is an important concept in quality assurance
programme. An effective QA procedure should allow an
audit trail to be followed back from the laboratory report.
This should allow access to a complete documented history
from receipt of the specimen to the issue of the report.
Documentation
Document is a record of any information or instructions
including policy statements, quality manuals, procedures,
specifications, calibration tables, reports, job description,
documents of external origin such as regulations, standards
Training
The quality system is only as good as the staff who actually
work with it. No matter how good the quality system is on
paper, if the theory cannot be translated into practice,
quality cannot be achieved. Training must also include an
understanding of why quality is important. Training should
be competency based and must be followed by a post-
training support to provide a continuous support.
Existence of a quality system demonstrates that the
laboratory has:
â commitment to quality
â a definite programme for quality and its
continuous improvement
â methods for processing laboratory specimens in
the form of approved written SOP
â evidence based control systems
â appropriate documentation
â trained human resource
â mechanism for error management under which it
can detect when and where things have gone
wrong and take necessary actions to prevent
recurrence of such episodes
Development and
Procedures Ú application of SOP
Methodology to carry
Work instructions Ú out specific jobs
Implementation of quality
Training of staff Ú system and use of SOP
5.1.1 Staff
The staff in peripheral laboratories should include at least
one technician and one laboratory assistant/attendant.
5.1.2 Space
Space available in peripheral laboratories should include at
least one laboratory-cum-office/record room (approx. 5
metres x 3 metres ) and one store-room which can be used
for other services also (approx. 5 metres x 3 metres).
5.2.1 Staff
Qualified pathologist/ microbiologist 1
(Doctor of Medicine/diploma in clinical
pathology)
Technicians - DMLT (diploma in medical 2
Laboratory technology) with experience
Laboratory Assistants (DMLT) 1
Laboratory attendants 2
Cleaner 1
Clerk-cum-storekeeper 1
Since it may not be possible to have a full-time
epidemiologist, services of an epidemiologist should be
available at least on part time basis.
5.2.2 Space
Microbiology/Serology laboratory 1
(approx. 8 meters x 5 meters)
Sterilization, media preparation laboratory 1
(approx. 6 meters x 4 meters)
Store-room (approx. 3 meters x 5 meters) 1
Office (approx. 3 meters x 5 meters) 1
5.2.3 Equipment
Binocular microscope 2
Dark-field microscope 1
Inoculating chamber 2
Centrifuge 2
Autoclave 2
Incubator 2
Hot air oven 1
Water bath 2
VDRL shaker 1
Colorimeter 1
Refrigerator 1
Balance 2
pH meter 1
Inspissator 1
Distilled water apparatus 1
Micropipettes as per
workload
Tips for pipettes as per
workload
The tests expected to be performed at the
intermediate laboratories are listed in Table 5.2.
For detection/
Procedure/Specimen
diagnosis of
Urethra/vaginal exudate Gonorrhoea
Wounds/pus Clostridia/other
organisms
Cultures
Nasopharyngeal specimen Corynebacterium
diphtheriae,
Streptococcus
pyogenes
Sputum AFB, cocci, others
CSF AFB, cocci,
Haemophilus
influenzae
Exudate/pus Bacterial infections
Blood Salmonella typhi,
other salmonellae;
Brucella Streptococci,
Meningococci,
Haemophilus
influenzae
Gastric washing AFB
Urethral/vaginal exudate Neisseria gonorrhoeae
Chlamydia
Haemophilus ducreyi
Faeces Salmonella and
Shigella
Vibrio cholerae
Escherichia coli and
others
Food poisoning
bacteria
For detection/
Procedure/Specimen
diagnosis of
Urine Pyogenic organisms
and AFB
Antibiotic sensitivity For various pathogens
Serological tests
Widal Enteric fever
Tube agglutination Brucellosis
ASO Rheumatic fever
VDRL Syphilis
Rapid diagnostic tests for Detection of HBsAg for
hepatitis B, HIV
antibody,
Meningococcal
antigen
KOH preparation of Fungi
skin/nail/hair
Environmental samples Bacteriology of water
and food
Policy/Plan 1
2 Manual
SOP 3
4 Worksheet, form
form record
record
(1) The latest version is at the work place. This means that
the latest information/procedure can be used/followed
by the staff performing a specific activity;
â Work sheets
â Reagent specifications
â List of approved suppliers
Documents such as forms, patients records etc. need
not be controlled.
Format
Most records are paper forms that are manually completed
by personnel. However, modern technology has also
brought in the use of computers for better storage and
retrieval of data. Computers can also be used for generation
of hard copies for sending to the user as well as compiled
data in the form of reports to supervisors or regulatory
agencies.
Retention of Records
There are no hard and fast rules regarding the period for
which records should be retained in laboratories. In
general, records are kept for two years, except for recording
and reporting instruments and equipment which have to be
kept and maintained for the life of the equipment.
Irrespective of the period of retention, the storage should
be such that it permits easy access for review, as required.
Accession List
This list records all specimens that are received in the
laboratory for processing. The information to be included in
this list is given below:
â Name of patient
â Identifying number (OPD No. / Admission No )
â Specimen type and source
â Name of test requested
â Date of collection (if possible time of collection
also)
â Time and date of receipt in the laboratory
â Specimen accepted or rejected. If rejected, reason
for same, e.g.
− improper labelling
− improper collection
− improper transport of specimen
Requisition Form
The requisition form is the record on which the test of the
specimen requested by the patient’s attending doctor is
recorded. A suggested list of information in the requisition
form, which obviously has to be separate for each
specimen, is provided below:
â Name, age and sex of patient
â Identifying number (OPD and/or admission
number)
â Location of patient (Ward and bed number)
â Presumptive diagnosis with clinical notes
Work Card
A work card is assigned to each specimen so that
procedures performed on the specimen, notes by the
laboratory personnel, results obtained and interaction
between the physician and the laboratory staff can be
recorded. A properly completed work card can be used to
reconstruct and assess the accuracy of the final report. It
should contain the following information:
â Name, age and sex of patient
â Identifying number (OPD and/or admission
number)
â Specimen type and source
â Name of test requested
â Initials of laboratory personnel performing
procedures
â Procedure, date performed and media used
â Preliminary results with
− direct microscopy
− culture
− colony characters
− biochemical and other tests for identification
â Final diagnosis
â Antimicrobial susceptibility and resistance
â Any discussion with physician
Test Report
Test reports convey the laboratory data to the doctor
requesting the test. These must be unambiguous and
precise and become part of the permanent hospital record
of the patient. The desired information in these reports
include:
â Name and location of patient
â Identifying number (OPD and/or admission
number)
â Name of physician requesting the test
â Specimen type and source
â Name of test requested
â Date specimen received
â Date specimen processed
â Name of laboratory personnel performing the test
and reporting
There are four types of reports that can be sent out
from the laboratory.
Telephonic report: Critical information that may be
urgently required by the physician should be reported by
telephone, followed by a proper report on proforma.
Positive microscopic smears, antigen tests, detection of
growth from normally sterile sources or detection of highly
infectious diseases should be communicated by telephone.
Preliminary report: This indicates the status of test results
within 24 to 48 hours of receipt of the specimen. If a
Incident Reports
Incidence reports document problems related to the
performance of care givers (e.g. collection, labeling or
transport of specimen; complaints; uncooperative
behaviour of laboratory personnel in processing of
specimens, safety violations). The information that has to be
incorporated in these is as follows:
â Date
â Name of reviewer
â Date of review
Safety Records
Safety records document that all employees with a potential
occupational exposure to hazardous chemicals or
potentially infectious material have participated in a training
programme at the time of employment and annually
thereafter. This document should have following
information:
â Dates of training sessions
â Contents of training sessions
â Names of persons conducting training
â Names of persons attending training
ACCURATE
S
STRICT ADHERENCE
BY ALL
ENCOMPASS ALL
ACTIVITIES O
READILY AVAILABLE P
Details of procedures
Gives in detail the examination procedure indicating differential
tests, flow charts or keys as well as identification criteria.
Differential tests
Need description in detail regarding
– Title
– Principle
– Material ( e.g. colony to be tested )
– Reagents
(a) source
(b) preparation technique
(c) storage technique
– Standards and controls
– Directions for performing the test
– Interpretation of results
– Commonly encountered problems and their solution
Antimicrobial susceptibility testing
Organism that can be tested and the antimicrobial agent which is
to be used for testing.
Serological testing
Includes details as given above under heading differential tests.
Reference to higher laboratories
How to use the reference laboratories
All staff must understand the limitations of his laboratory and
quickly despatch relevant samples to the Reference Laboratory
with a request for appropriate tests for which the forwarding
laboratory is not well-equipped.
Quality control
Including the laboratory’s written policy stating time and
frequency for performing quality control steps for media, reagents,
antibiotic discs, sera. Instructions must state what control results
are acceptable and what results are not. It is also important to
specify how these results are to be recorded and the actions to be
taken when deviations occur.
Reporting
Including clear cut instructions about reporting results.
â Definitions
â Culture media and reagents/material
â Equipment
â Sampling
â Test procedure
â Results
â Calculation
â Quality assurance
â Reporting
â References
8.1 DEFINITION
ISO 9001 defines validation as the attaining and
documenting of sufficient evidence to give reasonable
assurance, given the current state of science and the art of
Process of validation
â Plan and define aims of validation
â Develop protocol for the process of validation
â Execute the process and collect data
â Compare results against agreed requirements
â Consider any other issues: e.g. health and safety
â Accept or reject
â Document
â Implement
â Review
Validation is that part of a quality system that evaluates
in advance the steps involved in operational procedures or
product preparation to ensure quality, effectiveness and
reliability. This is a tool that controls changes. Validation
ensures that when new process/equipment or software is
introduced it performs correctly thus ensuring that the
quality of the product is not compromised. In some
instances this may be necessary for regulatory requirements.
Monitoring Evaluation
Concurrent Retrospective
Continuous Periodic
9.1 MONITORING
Main objectives of monitoring are
â to confirm consistency
â to alert to change
â to assess the impact of changes to processes or
procedures
â to identify opportunities for improvement
â to provide objective measurements
The decision as to what to monitor depends upon the
work-area and the activities. It is really common sense what
is monitored, but it is usually best to start with parameters
that are easily measurable and have profound influence
upon the quality of the laboratory results.
This may mean starting with the final output of
processes rather than the intricate details, but that is
acceptable as long as the factors that influence the final
outcome are known.
Once the decision has been made to monitor and
what to monitor, the question is how to collect the
appropriate data and analyze the same.
Benefits of EQAS
â Helps laboratories in comparing their results with
other laboratories
â Acts as an educational stimulus to laboratory staff
â Participation provides credibility to the laboratory
â Helps the health administrators and regulatory
agencies to have an insight into the status of
quality across the country, identifying the
problems and devising methodology to overcome
these
Process of EQAS
EQAS requires a well equipped, experienced laboratory at
intermediate or central level to act as the organizing
laboratory and a fairly reasonable number of laboratories as
the participating laboratories. The process of EQAS with
Ú
Prepare Quality Assurance
Specimens Ú Examine Specimens
Requirements of EQAS
EQAS includes submission of samples to participating
laboratories; analysis by them and returning of the results to
the EQA organizer who performs the statistical analysis and
sends feedback to the participants so that they may judge
their individual performances. The essential requirements
are briefly discussed below:
Principles
There can be different principles guiding the organization of
EQAS. In some schemes, specimens are sent out every
month to the participants; in other schemes they are sent
out every three or six months. Some schemes check the
precision only; others check for precision and accuracy.
Some schemes send out two specimens (usually one
specimen with values in the normal range and the other in
the pathological range). Whatever principles are adopted
by the organizers should be related to the participants in
unambiguous terms.
Frequency of Distribution
The frequency of distribution of quality assessment
specimens in an EQAS is a matter of policy. There are
schemes in Europe that have only two surveys per year,
each survey with two specimens for investigation. Other
EQAS have 3,4,6 or 12 surveys per year. Each of these has
its advantages and disadvantages (from the educational,
organizational and economical point of view). EQAS with
12 surveys are usually more expensive. A frequency
distribution should be chosen which makes possible good
preparation of the materials to be distributed and careful
analysis of the results from the laboratories.
any possible errors they may have made. In this way the
EQA will also have an educational function.
The presentation of the analysis of results should be
clear and precise so that the participants can easily judge
the quality of their work and can correct errors wherever
necessary.
EQAS process
A summary of the process from when the decision is made
to set up an EQAS to the time a report is produced and
distributed to participants involves not only planning,
research, evaluation and validation etc, but the actual
process of designing, manufacturing, distributing the panel
then collecting, collating and analysing the results and
sending out a report before considering the appropriate
feedback.
The steps involved include:
Applications of IQA
The applications of IQA include
â Assessment of variability in tests where subjective
interpretation plays an important role
â Statistical analysis and confidence limits on repeat
testing
â Assessment of effect on changes in procedures or
introduction of a new technique
For internal quality assessment (IQA), the specimen is
split in the laboratory and one half is processed as a
patient’s specimen and the other half by the same protocols
as a known IQA sample. This provides a measure of
precision and throws light on the effectiveness of the quality
system.
Definition
Quality audit is defined as a planned and documented
activity performed in accordance with written procedures
and check-lists to verify by investigation, and the
examination and evaluation of objective evidence, that
applicable elements of a quality assurance programme have
Fire
Fire is a potential hazard in almost any environment.
Instructions for emergency action in case of fire must be
prominently posted and must be required reading by all the
employees. Some of the important preventive measures are
described below.
â Prominent display of emergency directions
â Fire drills, some of which may be without warning
â Distinct marking of fire exits
â Fire exits kept unblocked
â Fire extinguishers: Available, functional and staff
trained in their use
Chemical Hazards
The use of hazardous chemicals in the laboratory can cause
serious injury to workers if they are not instructed and
trained in how to handle them safely. Overt exposure can
result in serious tissue damage and acute adverse effects.
Safety goggles should be worn when there is a potential
hazard of chemical splashes. Various other preventive
measures which should be instituted are given below:
Electrical Hazards
Some electrical units can cause electric shock and fire
hazards. Explosions in electrophoretic units causing fires
have occurred frequently. The microwave oven has
become a common piece of laboratory equipment, but its
use has introduced a significant new hazard to the
laboratory. Careless operating procedures have resulted in
violent release of superheated fluids and explosions caused
by rapid pressure build up in accidentally sealed containers.
Serious injury is always a potential consequence of the
misuse of this equipment. To prevent these all electrical
equipment should be earthen (grounded); information
regarding location of master switch be prominently posted
and uninterrupted source of power should be provided.
Microbiological Hazards
Microorganisms will invariably be found in microbiology
laboratories. The greatest risk of occupational infection in
these laboratories is associated with the use of pathogenic
microorganisms or the handling of contaminated material.
The ability to prevent laboratory-acquired infections
requires skill and knowledge which can best be acquired
through training and careful guidance. It is necessary that
proficiency in microbiological techniques be acquired
through practice with nonpathogenic microorganisms
before higher-risk microorganisms are introduced into the
Level 1 Biosafety
Level 1 biosafety is not appropriate for work with
pathogenic bacteria. This is applicable to basic practices
appropriate for undergraduate and secondary educational
training and teaching laboratories for which introductory
microbiological protocols would involve only defined and
characterised strains of viable microorganisms not known to
cause disease in healthy adult humans. Emphasis is placed
on:
â the use of mechanical pipetting aids
â handwashing
Level 2 Biosafety
Level 2 biosafety practices are obligatory for most
laboratory activities involving known pathogens and for
experiments involving either the introduction of
recombinant DNA into pathogenic bacteria or the
introduction of DNA from pathogenic bacteria into
nonpathogenic prokaryotes or lower eukaryotes.
Laboratory Access
â Appropriate signs should be located at points of
access to laboratory areas directing all visitors to a
receptionist or receiving office for access
procedures.
â The universal biohazard symbol (Figure 11.1)
should be displayed at specific laboratories
handling pathogenic microorganisms. Only
authorized visitors should enter a laboratory
displaying the universal biohazard sign. Doors
Clothing
â All employees and visitors in microbiological
laboratories should wear protective laboratory
clothing and shoe covers.
â Disposable gloves should be worn wherever
microbiological work of moderate to high risk is
undertaken (e.g. processing of blood samples for
HBsAg and HIV testing).
â Laboratory clothing including shoes should not be
worn outside the work area.
â Clothing from laboratories in which moderate and
high risk virus manipulations are being performed
should be autoclaved before sending these to
laundry.
Pipetting
There should be strictly no mouth pipetting in any laboratory.
Housekeeping
â Dry contaminated wastes from laboratories should
be collected in impermeable bags which should be
sealed at the collection site before removal to the
autoclave or incinerator. Metal cans with tight
sealing covers may be used in lieu of bags. The
Storage
Prior to disposal, all biohazardous waste should be
maintained and stored separately from the general waste
stream and from other hazardous wastes. The containers
used to store biohazardous waste should be leak-proof,
clearly labeled with a red or orange universal biohazard
symbol and sealed tightly when transported. In certain
cases it may be necessary to double-bag the waste to
prevent leakage. Any biohazardous sharps, such as
infectious needles and scalpels, must be placed in
containers that are puncture resistant, leak proof on all
sides and the bottom, and closable. These containers can
then be placed in a standard biohazard bag.
Disposal options
There are three main disposal options:
â render waste noninfectious by autoclaving and
dispose it in the general waste stream
â on site incineration, if possible
â transportation of locally generated waste to a
distant appropriate facility
Incineration is the preferred disposal option. Not only
does this method render the waste noninfectious but it also
changes the form and shape of the waste. Sterilization is an
effective method for decontaminating waste, but it does not
alter the appearance of the waste. Steam sterilization in an
autoclave at a temperature of 121°C for at least 15 minutes
destroys all forms of microbial life, including high numbers
of bacterial spores. This type of complete sterilization can
also be accomplished using dry heat which requires a
temperature of 160-170°C for 2-4 hours. However, it must
be ensured that heat comes in contact with the material to
be rendered sterile. Therefore, bottles containing liquid
material should have loosened caps or cotton plug caps to
Parameter Guidelines
Document routine preventive maintenance
Retain maintenance records for life of equipment
Commercially prepared Inspect each shipment for cracked media or petri
media dishes, haemolysis, unequal filling, excessive bubbles
and contamination
Document deficiencies, take corrective action, inform
manufacturer
Perform in house QC testing
User prepared media Record amount prepared, source, lot numbers,
sterilization method, preparation date, pH, expiration
date.
Check medium for colour, consistency, depth or slant,
smoothness, haemolysis, contamination, bubbles
Test media with QC microorganisms of known
characters
Stains, reagents and sera Label containers as to contents, concentration, storage
requirements, date prepared, received/ placed in
service, and shelf life
Store as per recommendations
Test with positive and negative controls prior to use
Discard outdated materials and reagents that fail to
perform
Commercial kits Test each batch as per recommendations of
manufacturer
”
Label appropriately
”
Rapidly transport to laboratory in unchanged state
”
Proper accession in laboratory
”
Perform accurate and precise analysis
”
Document and report
”
Proper interpretation
”
Timely action on right patient
Westgard Rules
These detect both systematic and random errors. These
rules also define the specific performance limits. There are
six Westgard Rules of which three (12SD, 22SD and 41SD) are
known as warning rules and three (13SD, R4SD and 10x) as
mandatory rules. The violation of warning rules suggests a
review of test procedures, reagent performance and
equipment calibration. Any deviation from the mandatory
rules requires rejection of results on patient’s material in
that assay. These have been summed up in Table 12.3.
Cumulative sum
Cumulative sum (CUSUM) is the method for identifying
shifts or trends occurring in an assay. It indicates systematic
errors which may include deterioration of reagents or poor
equipment calibration. The established target value and SD
of the QC samples are used to determine the upper and
lower threshold limits for the controls and set at +/- 1SD.
Any value outside this range should trigger calculation of
CUSUM.
13.1 PIPETTES
Pipette, the most frequently used volumetric glassware, play
a crucial role in determining the degree of accuracy and
precision in a great many analyses performed in the
laboratory. The greater the number of manual test methods
used in the laboratory, the greater the role played by the
pipettes. Completely automated laboratories do little or no
pipetting at all. In any case, if pipettes are used in the
laboratory, these should be properly selected and used with
care.
13.4 CHEMICALS
American Chemical Society’s (ACS) Committee on
Analytical Reagents has established specifications for
chemicals to qualify as analytical reagent (AR) grade. AR
chemicals are of very high purity and, whenever available,
should be used in all analyses in the clinical laboratory.
Manufacturers of these chemicals analyze their product and
label them AR or ACS only if they meet ACS specifications.
AR chemicals are analyzed for purity, which is usually
marked on the bottle. The impurities are also analyzed and
reported along with the purity. The purity of the chemicals
is also indicated by terms such as p.a. (pro analysis) and
p.p.a. (purissimum pro analysis).
The use of AR chemicals in the laboratory cannot be
overemphasized. No one wants to analyze chloride in
serum with a reagent already containing chloride or
magnesium with a chemical containing magnesium.
Dehydrated Media
These are commercially available and require only the
addition of water to be reconstituted for use. The
responsibility for quality control lies with the manufacturer.
However, it has to be tested for its quality, after
preparation, because of changes that can be brought about
by the process of reconstitution and sterilization.
Water
Measure carefully the amount of water that is added when
reconstituting media. Since impurities render tap water
unsuitable for the preparation of most biological media,
laboratories should use either distilled water, deionized
water, or water that has been treated in both ways.
Weighing
Accurate balances should be used for weighing dry
materials. Weighing errors significantly alter the
composition of the final product.
Dispensing
Media should be dispensed accurately and aseptically in
plates and tubes. Failure to measure the amount accurately
may result, for example, in too shallow or too deep agar
medium, either of which may make the medium unsuitable
for use.
Proper Sterilization
A common error in media preparation is sterilizing media at
too high a temperature or for too long a period, or both.
This may result in deterioration or decomposition of some
constituents of the media, which will render the media
useless for the intended purpose.
Glassware
Care should be taken to use clean glassware, since residues
on glass may be inhibitory to some fastidious micro-
Physical Appearance
If the medium is stored for an excessively long time under
adverse conditions or has been improperly prepared, the
following signs may develop and these should be
documented.
â Presence of turbidity or a precipitate indicates that
some constituent has come out of the solution.
â Colours darker than normal may indicate
overcooking of sugar containing media, incorrect
pH or incorrect mixture of ingredients.
â Colour lighter than normal may also indicate
incorrect mixture of ingredients or a wrong pH.
â Prolonged storage of medium after pouring in
plates causes its dehydration and makes it unfit for
use. Dehydration of the medium can be reduced
by preparing only required number of plates of
Sterility
A few media are used without terminal sterilization, but
these are exceptions; most media must be sterile when they
are inoculated. Each batch of medium, whether prepared
in the laboratory or received from a commercial source,
should be sampled for sterility. This is best done by
removing 1-5% of the batch and placing it in a bacteriologic
incubator at 35°C for 48 hours. If contaminants appear in
the medium as a result of inadequate sterilization, a new lot
should be obtained. Those containers that are used for
sterility testing should be discarded at the completion of the
test, since they are unsuitable for inoculation because of the
dehydration that occurs after up to 48 hours in the
incubator.
Growth
Determine the ability of the medium to support the growth
of suspected organisms by inoculating the medium with a
typical stock culture isolate. A frequent quality control error
is the use of a heavy inoculum for this purpose. For most
media, inoculating with a stock culture that is too heavy
may result in misleading growth. In a specimen, the
organism may be much more fastidious or present in very
small numbers; therefore, the medium may not support its
growth. When testing for the ability to support growth, it is
good to prepare a dilute suspension to use as the inoculum.
This suspension will give greater assurance that the medium
is adequate for the growth of a small number of organisms
in a patient’s specimen. In selecting an organism for testing,
one should select from among the more fastidious species
Biochemical Response
When inoculating media used to identify a specific
reaction, such as fermentation or H2S production, it is
necessary to use only a species or strain of organism that
will produce the desired reaction.
Selective Media
Since selective media are designed not only to support the
growth of organisms but to inhibit the growth of others, it is
necessary to inoculate the medium with representatives of
both groups of organisms. To demonstrate the inhibitory
effect, one can challenge the medium with a heavy
inoculum, since, if the medium will prevent the growth of a
large inoculum, it will inhibit the small number of organisms
that may be present in the primary specimen. The medium
must also support the growth of the selected organisms.
As a matter of general principle, each batch of culture
medium should be checked before use with control strains
to ensure that it supports the growth of bacteria and, in the
case of selective media, inhibits the growth of undesirable
organisms. However, if economics does not permit this
approach, those media which are known from experience
to be trouble free and reliable need not be subjected to
such a regular quality control regimen. The laboratory has
to identify such reliable media and accordingly establish
quality control schedules. This concept must be periodically
reviewed. However, whenever a new batch of medium,
new supplier or a new product is to be used it is prudent to
Procedure/ Expected
Control organism Expected reaction
Test result
Bacitracin Streptococcus + Zone of inhibition
disc group A – No zone of inhibition
Enterobacter
faecalis
Optochin Strept. Pneumoniae + Zone of inhibition
disc Strept. Viridans – No zone of inhibition
ONPG Esch. Coli + Yellow colour
disc Proteus vulgaris – No change in colour
Oxidase P aeruginosa + Purple colour in
disc Esch. Coli – 30 seconds
No change in colour
It is also essential to undertake quality control
procedures at regular intervals. These should be performed:
â With each new batch of reagents
â With each new vial of reagent
â Daily for catalse, oxidase, and coagulase
â Weekly for bacitracin, optochin and ONPG
A test procedure not giving anticipated results with the
control organisms should not be used till such time that
remedial steps have been taken to correct the problem.
â prevent contamination
â ease of supply and transport of preserved
microorganisms
â duration of preservation
â cost
Various methods of preservation and their advantages
vis a vis the factors described earlier are shown in Table
16.1
Subculture + + ++++ + + +
Gelatin ++ ++ + + ++ ++
discs
Lyophili- +++ +++ + +++ +++ +++
zation
Enterobacteriaceae
Escherichia coli (ATCC 25922) is required as one of the test
organisms in performing antibiotic susceptibility testing by
the Kirby-Bauer technique. Many members of family
enterobacteriaceae may also be employed for checking
various differential media such as MacConkey, eosin-
methylene blue agar (EMB), xylose-lysine-desoxycholate
agar (XLD), triple sugar iron agar (TSI), potassium cyanide
medium (KCN), and methyl red/Voges-Proskauer broth
(MR/VP), as well as different biochemical tests. If the
laboratory is required to test for enteropathogenic Esch. coli,
it is also desirable to maintain some of the cultures of the
recommended standard strains for checking the activity of
the immune serum to the most commonly encountered
types.
All members of the family enterobacteriaceae are
readily maintained on heart infusion agar slants under oil at
room temperature with yearly transfers.
Pseudomonas
Pseudomonas aeruginosa and P. maltophilia are useful for
checking oxidation-fermentation (OF) media and Seller’s
medium. The organisms may be maintained on heart
infusion agar slants under oil at room temperature with
yearly transfers.
Staphylococcus
Staphylococcus aureus (ATCC 25923) is required as one of
the test organisms for performing antibiotic susceptibility
testing by the Kirby-Bauer technique. In addition, it can be
used for checking plasma for the coagulase test; for
checking culture media for the production of indole,
catalase, and DNase; for the reduction of nitrates, and for
checking the Gram stain. Staph. epidermidis is used for a
negative control in the coagulase and DNase tests.
Streptococcus
Streptococcus pyogenes is necessary for checking bacitracin
discs, the quality of group A antisera, the ability to produce
β-haemolysis on blood agar plates, and to grow on blood
agar containing colistin and nalidixic acid.
Vibrio
Vibrio cholerae will grow easily on heart infusion agar.
Good growth takes place on heart infusion agar slants
containing 1.5% NaCl, and the slants may be held under oil
at room temperature.
Fungi
Fewer and less complex culture media are required for
fungi than are necessary for bacteria in a clinical diagnostic
laboratory, and so there are fewer requirements for quality
control. The necessary cultures of fungi may be maintained
on slants of Sabouraud’s agar in tubes with screw caps at
room temperature and in the dark. Transfers need to be
made every 2 or 3 months. Sterile mineral oil may be
added to the slants after the cultures have attained their
optimum growth, and then transfers may be made.
Set 1 Set 2
Chloramphenicol
Co-trimoxazole
Intestinal Nalidixic acid
Tetracycline
Sulfonamide Norfloxacin
Trimethoprim Chloramphenicol
Enterobacteriaceae Co-trimoxazole Gentamicin
Urinary Ampicillin
Nitrofurantoin
Nalidixic acid
Tetracycline
Ampicillin Cefuroxime
Chloramphenicol Ceftriaxone
Co-trimoxazole Ciprofloxacin
Blood and tissues Tetracycline Piperacillin
Cefalotin Amikacin
Gentamicin
Piperacillin Amikacin
Pseudomonas
Gentamicin
aeruginosa
Tobramycin
Antibiotic Discs
Any commercially available discs with the proper diameter
and potency can be used. Stocks of antibiotic discs should
preferably be kept at -20oC; the freezer compartment of a
home refrigerator is convenient. A small working supply of
discs can be kept in the refrigerator for upto 1 month. On
removal from the refrigerator, the containers should be left
at room temperature for about l hour to allow the
temperature to equilibrate. This procedure reduces the
amount of condensation that occurs when warm air reaches
the cold container.
Turbidity Standard
Prepare the turbidity standard by pouring 0.6 mL of a 1%
(10 g/L) solution of barium chloride dihydrate into a l00-mL
graduated cylinder, and filling to l00 ml with 1% (10 ml/L)
sulfuric acid. The turbidity standard solution should be
placed in a tube identical to the one used for the broth
sample. It can be stored in the dark at room temperature
for 6 months, provided it is sealed to prevent evaporation.
Swabs
A supply of cotton wool swabs on wooden applicator sticks
should be prepared. These can be sterilized in tins, culture
tubes, or on paper, either in the autoclave or by dry heat.
Procedure
â To prepare the inoculum from a primary culture
plate, touch with a loop the tops of each of 3-5
colonies of similar appearance of the organism to be
tested.
Yes No
Control Limits?
No
No Yes
No Yes
Standard Strains
These are:
â Staphylococcus aureus (ATCC 25923)
â Escherichia coli (ATCC 25922)
â Pseudomonas aeruginosa (ATCC 27853)
Culture for day-to-day use should be grown on slants
of nutrient agar (tryptic soya agar is convenient) and stored
in the refrigerator. These should be subcultured onto fresh
slants every 2 weeks.
Haemolysed Blood
Haemolysed blood specimens are not suitable for
serological studies. It is always advisable to avoid factors
which cause haemolysis (Table 18.1). Specimens containing
precipitates should be centrifuged prior to testing.
Preparation
Sera to be used as controls should be kept sterile to avoid
deterioration. In general each procedure should have a
normal control serum (negative), a strong positive control
serum, and another positive control serum which is reactive
at the critical concentration (borderline positive). With
some tests, controls with a low concentration of analyte
should be included. Controls recommended by the
manufacturer of a particular test should always be used and
additional control sera can be included if a test involves
special problems.
Storage
Sera to be used as controls should be standardized against
international reference materials when they are available.
“Standards” included in commercial kits are not calibrated
with each other and often are not interchangeable. These
should be stored in aliquotes in frozen forms. Repeated
freezing and thawing should be avoided.
18.5 REAGENTS
Quality reagents are necessary for quality performance. A
record should be kept of any changes in reagents in case
Biological Indicators
Bacillus stearothermophilus was earlier considered ideal for
monitoring because this organism lacks pathogenicity,
pyrogenicity and toxicity. Biological indicators
manufactured today are generally impregnated with a spore
population to meet a performance requirement of surviving
a certain period of time in a sterilizing atmosphere but
being killed in a longer period of time at the same sterilizing
conditions. The number of spores that should be present
when sterilization is being monitored is 104 to 106 for
B.stearothermophilus and around 106 for Bacillus subtilis var
niger.
Heat Sterilization
For heat sterilization, the first requirement is knowledge
that the temperature is recording correctly. Furthermore,
the operator must know that the temperature is reaching all
the parts of the load and is maintained for the desired
length of time. Recording thermometers and barometers for
steam sterilizers should be employed for the chambers and
thermocouples can be buried inside the load. Paper strips
treated with chemicals that change colour at the required
temperature may be used. If, in a steam autoclave, a
container is tightly closed and receives no steam, it may
reach the correct temperature, but this will not ensure that
sterilization will occur. To give this assurance requires the
use of biological testing in the form of heat-resistant spores.
With moist heat, spores of B. stearothermophilus are used,
and with dry heat sterilizers, spores of B.subtilis var niger
are selected. The spores are dried on paper treated with
nutrient medium and chemicals. After the sterilization
treatment, they are incubated for germination and growth
and a colour change indicates whether they have or have
not been activated. This method may take several days of
incubation, whereas physical and chemical methods are
immediate, but the biological tests are more dependable.
Chemical Sterilization
For chemical sterilization, there are colour indicator tapes
for ethylene dioxide and formaldehyde, which show
whether or not these gases have penetrated in sufficient
quantity at the prescribed temperature to provide
sterilization. But here, as with other methods, biological
methods are preferable. With ethylene oxide, strips treated
with B.subtilis var niger are employed, whereas with
formaldehyde B.stearothermophilus is used.
Sterilization by Filtration
With filtration sterilization, membrane filters may be readily
tested for passage of microorganisms of different sizes.
Spores of B.subtilis var niger, cells of P. diminuta,
bacteriophages and other viruses give a range of sizes. The
bubble point test can also be used. This correlates the pore
diameter with the air pressure required to cause the first
bubble to break through a filter. Depth filters may be tested
by the passage of selected organisms or by the penetration
of aerosols made up of chemical dusts of known particle
size. For example, dry particles of sodium chloride can be
detected and quantitatively determined with a hydrogen
flame photometer.
Combined Treatments
Enhanced sterilizing activity can take place if two or more
processes, chemical or physical, are employed together.
There are various kinds of such treatments as discussed
below:
Thermochemical Treatment: With an increase in
temperature the antimicrobial activity of various
Glass Paper
slides cups
syringe and needles plates
Wood tubes
tongue depressors Rubber tubings
applicator catheters
Steel tumbler drains
Autoclave
An autoclave is an integral part of any microbiological
laboratory. Various steps that can be taken to ensure its
proper functioning are provided below:
Incubators
Incubators should be subjected to continuous recording of
temperature. However, if it is not possible, the temperature
must be recorded every day and before opening of the
incubator.
pH Meter
A pH meter needs to be standardised before each run with
a standard buffer of pH 7.0. However, in instances when
the work is related to a pH range of less than 6.0, it is
advisable to use a standard buffer of pH 4.0. The buffer
solution should be checked monthly with another pH meter
and discarded if the pH deviates more than ± 0.4 or if the
buffer is contaminated with microorganisms.
Centrifuge
A centrifuge should be evaluated often enough to assure
proper performance. The rheostat control should be
checked with a tachometer at various loadings and at
frequent intervals (e.g. six monthly) to assure proper
gravitational fields.
Volumetric Glasswares
All volumetric glassware such as flasks and pipettes, should
be checked for proper calibration before being used. All
glassware must be clean and free of detergents. Chipped or
etched glassware should be discarded.
Pipette
Each pipette, whether manual, semiautomated or
automated, must be tested periodically to determine if it is
delivering the correct volume.
Timers
Mechanical timers should be tested on a regular basis to
determine their accuracy by comparing them with
electronic or electric clocks.
20.3 DOCUMENTATION
The assurance that a piece of equipment is operating
properly can best be judged by examining its performance
over time. Records of performance parameters, therefore,
are a vital element in the proper operation of laboratory
equipment. Some suggested information is provided below:
â Name and serial number of instrument
â Elements to be checked and kind of data to be
collected
â Frequency of checking
â Record of data
â Comments on data
â Changes made to restore accuracy and precision,
if any
â Signature with date of the person performing these
tasks
Snell JJS, Farell ID, Roberts C. Eds Quality control: principles and
practice in the microbiology laboratory. 2nd Edition
(Public Health Laboratory Services, Colindale, London), 1999.
Taylor, RN, Huong AY, Fulford KM, Przybyszewski VA, Hearn TL.
Quality control for immunologic tests
(CDC, Atlanta Ga), 1985.
Monitoring, 62
Objectives of quality assurance
Total quality management, 18
Peripheral laboratory services, 27
Practice of laboratory safety, 88
Precision, 163
Preservation of stock cultures, 136
Preventive maintenance, 182
Quality
Assessment of, 62
Factors affecting analytical, 8
Pre-analytical factors, 9
Post-analytical factors, 13
Quality assurance, 17
Quality assurance programme,
Statistical challenges, 112
Components 19
Computers 20
Quality assurance in antibiotic susceptibility testing, 141
Factors influencing zone size in, 152
Troubleshooting guide for disc diffusion, 153
Quality assurance in clinical laboratory, 107
Quality assurance programme, 17
Quality audit, 80
Quality control
equipment, 177
serology, 160
sterilization, 170
bacteriological media, 133
stains, 132
Quality system, 21
Key elements, 22
Rapid diagnostic tests, 36
Referential (quality) standards, 23
Requisition form, 45
Safety in the laboratory, 87
Safety records, 50